In recent years, reactive oxygen species (ROS) have been shown to have critical roles in normal vascular function and the pathogenesis of vascular disease. These molecules have profound effects on vascular smooth muscle cell (VSMC) growth, migration and differentiation. A major source of ROS in vascular cells is the NADPH oxidase (Nox) family of enzymes. Vascular smooth muscle cells (VSMCs) from large arteries express two types of NADPH oxidases, Nox1 and Nox4. These two oxidases are differentially localized within the cell, have distinct agonist specificity, and regulate specific cellular functions. What is less clear, however, is how the activity of these two enzymes is specifically regulated. While Nox1 regulatory mechanisms are similar to those of the phagocyte oxidase, Nox4 does not require any of the previously identified cytosolic oxidase regulatory subunits for its enzymatic activity. We have identified and cloned a new protein, NoxR1, that physically and functionally interacts with Nox4. Preliminary experiments indicate that NoxR1 overexpression in VSMCs causes a significant increase in NADPH oxidase activity, an increase in focal adhesions, and an increase stress fiber formation, while knockdown of NoxR1 induces a profound alteration of the cytoskeleton and impairs VSMC migration. The overall goal of this project is thus to define the physiological function of NoxR1 regulation of Nox4 and to determine its role in an in vivo model of migration. In the first specific aim, we plan to determine the role of NoxR1 in the regulation of focal adhesion turnover and cell migration, while Aim 2 is designed to determine the role of NoxR1 in neointimal formation in vivo using a newly created NoxR1 knockout mouse. Because NoxR1 is the first known regulator of Nox4, a Nox family member that regulates such basic cellular processes as senescence, differentiation and survival, these investigations are likely to have far-reaching implications for a number of vascular and nonvascular diseases. Modified Speracts with Nox4. Preliminary experiments indicate that NoxR1 overexpression in VSMCs causes a significant increase in NADPH oxidase activity, an increase in focal adhesions, and an increase stress fiber formation, while knockdown of NoxR1 induces a profound alteration of that therapy was begun too late in the disease process, and improper targeting of specific ROS (5). A better therapeutic approach is to prevent the pathological production of ROS by targeting the enzymes responsible for their formation. Of these, the NADPH oxidase family of enzymes is an important component. NADPH oxidases are multi-subunit enzymes that are ubiquitous, but highly regulated. Each cell type in the vascular wall expresses multiple NADPH oxidase homologues that have unique subcellular distributions, functions, and regulation. Vascular smooth muscle cells (VSMCs) from large arteries express two types of NADPH oxidases. Nox1 is comprised of the catalytic subunit (Nox1), its membrane associated partner p22phox and three regulatory components: p47phox, NoxA1 and Rac (6). Nox4, in contrast, has been shown to require p22phox for activity, but is not associated with any of the known regulatory components (7). While some reports suggest that Nox4 activity is constitutive, a number of investigators have shown that it can be activated by insulin (8), serum withdrawal (9), and TGF-[unreadable] (10). The functional role of Nox4 is still under investigation. It has been linked to senescence (11), growth (12-14), proinflammatory responses (15, 16), oxygen sensing (17), migration (18) and differentiation (9, 10, 19), suggesting that Nox4 must regulate fundamental cellular processes such as gene expression and/or cytoskeletal remodeling. Virtually nothing is known about the mechaases.